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                                Exploratory experiments
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                                Antipseudomonal drug production module
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                                C-di-GMP signaling and BC film production module
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                                Safety and drug release module</p>
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            <div class="content"
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                <div id="content1" class="yj">
                    ● Exploratory experiments
                </div>
                <div class="ej">1. MIC values</div>
                <div class="pb">Firstly, we tested the minimal inhibitory concentrations (MIC) of 4 antibiotics against
                    growth of <i>Gluconacetobacter hansenii</i> ATCC 53582, and the result was detailed in Table 1. The
                    MIC
                    data here would be employed in the following experiments.

                </div>
                <div id="tableHIde">
                    <table border="1"
                        style="font-size: 150%;text-align: center;line-height: 28px;margin-left: 395px;margin-top: 50px;">
                        <tr>
                            <td>Number</td>
                            <td>Antibiotics</td>
                            <td>MIC(ng/mL)</td>

                        </tr>
                        <tr>
                            <td>1</td>
                            <td>Gentamicin</td>
                            <td>300</td>
                        </tr>
                        <tr>
                            <td>2</td>
                            <td>Kanamycin</td>
                            <td>180</td>
                        </tr>
                        <tr>
                            <td>3</td>
                            <td>Chloramphenicol</td>
                            <td>148</td>
                        </tr>
                        <tr>
                            <td>4</td>
                            <td>Ampicillin</td>
                            <td>100</td>
                        </tr>
                    </table>
                </div>

                <div class="tz">Table 1. MIC values of different antibiotics against <i>G. hansenii ATCC</i> 53582</div>


                <div class="ej">2. Culture exploratory experiment</div>
                <div class="pb">To identify the optimal culture medium for <i>G. hansenii</i> ATCC 53582, an array of
                    growth
                    media
                    was selected for testing the bacterial growth. HS culture medium is commonly used for <i>G.
                        hansenii</i>
                    ATCC
                    53582, while HS-6, HS-7, and HS-8 are also utilized in some previous studies. Additionally, due to
                    the
                    similarity of medium composition to HS (Table 2), LB and SOC medium were included in our assay here.
                    Altogether, <u><span id="hidekey1" style="color: blue;">6 different bacterial growth
                            media</span></u> were employed to culture the
                    wild type
                    <i>G. hansenii</i> ATCC
                    53582 and J23100-mcherry-pSEVA331-<i>G. hansenii</i> ATCC 53582.

                </div>
                <div id="hideContent1">
                    <table border="1" style="font-size: 150%;text-align: center;line-height: 28px;">
                        <tr>
                            <td>Medium</td>
                            <td>Formula</td>

                        </tr>
                        <tr>
                            <td>HS</td>
                            <td>2% glucose,0.5% peptone,0.5% Yeast Extract ,0.68%
                                NaHPO<sub>4</sub>·12H<sub>2</sub>O,0.15%
                                Citric Acid Monohydrate </td>

                        </tr>
                        <tr>
                            <td>LB</td>
                            <td>LB Broth</td>

                        </tr>
                        <tr>
                            <td>SOC</td>
                            <td>SOC Broth</td>

                        </tr>
                        <tr>
                            <td>HS-6</td>
                            <td>8% glucose,1% peptone,0.2% Citric Acid Monohydrate,0.27%
                                NaHPO<sub>4</sub>·12H<sub>2</sub>O,0.03% MgSO<sub>4</sub>·7H<sub>2</sub>O</td>

                        </tr>
                        <tr>
                            <td>HS-7</td>
                            <td>8% glucose,2% YeastExtract,0.2% NaHPO<sub>4</sub>,0.1%
                                12H<sub>2</sub>OKH<sub>2</sub>PO<sub>4</sub>,0.02% MgSO<sub>4</sub>7H<sub>2</sub>O 1%
                                C<sub>2</sub>H<sub>5</sub>OH</td>

                        </tr>

                        <tr>
                            <td>HS-8</td>
                            <td>2% sucrose, 0.5% peptone,0.5% Yeast Extract, 0.68%
                                NaHP0<sub>4</sub>·12H<sub>2</sub>O,0.15%
                                Citric Acid Monohydrate</td>

                        </tr>


                    </table>
                    <div class="tz">
                        Table 2. Formula of 6 different bacterial growth media used in our assay
                    </div>

                </div>


                <div class="pb">Consistent with visual observation, the optical density of bacteria grown in HS, SOC and
                    HS-8 indicated bacterial growth, whereas bacteria in other media failed to grow, as illustrated by
                    low
                    or even negative values of OD<sub>600</sub>. In comparison, following 48 hours of culture, a higher
                    level of
                    biomass for bacteria grown in SOC, HS, LB and HS-8 was observed, with the highest OD<sub>600</sub>
                    observed in SOC
                    medium. Thus, SOC might be the optimal growth medium. Notably, OD<sub>600</sub> of bacterial culture
                    in
                    HS-8 at 48
                    hours was lower than that in 24 hours, an indicative of transition into the death phase in HS-8 at
                    this
                    timing. <strong>Taken together, based on the growth assay using a range of culture media, SOC and HS
                        were
                        opted
                        for the further experiments concerning <i>G. hansenii</i> ATCC 53582.</strong></div>


                <image src="https://2021.igem.org/wiki/images/f/f7/T--SZPT-CHINA--Results-pic-1.png" class="pic"
                    style="width: 1100px;"></image>

                <div class="tz">Figure 1. (a) Liquid culture of J23100-mcherry-pSEVA331-<i>G. hansenii</i> ATCC 53582
                    after
                    24
                    hours of agitation, (b) liquid culture of the wild type <i>G. hansenii</i> ATCC 53582 after 24 hours
                    of
                    agitation, (c) optical density of J23100-mcherry-pSEVA331-<i>G. hansenii</i> ATCC 53582 and wild
                    type <i>G.
                        hansenii</i> ATCC 53582 in different culture media after 24 hours of agitation, (d) liquid
                    culture of
                    J23100-mcherry-pSEVA331-<i>G. hansenii </i>ATCC 53582 after 48 hours of agitation, (e) liquid
                    culture of
                    <i>G.
                        hansenii</i> ATCC 53582 after 48 hours of agitation, (f) optical density of
                    J23100-mcherry-pSEVA331-<i>G. hansenii</i> ATCC 53582 and wild type <i>G. hansenii</i> ATCC 53582 in
                    different culture
                    media after 48 hours of agitation.
                </div>

                <div class="pb">
                    To quantify the bacterial cellulose (BC) production of <i>G. hansenii</i> ATCC 53582 in SOC and HS
                    medium, we
                    launched liquid culture of J23100-mcherry-pSEVA331-<i>G. hansenii</i> ATCC 53582 in HS and SOC
                    medium
                    with
                    different volumes (2mL, 2.5mL and 3mL) for 4 days. Then we measured the dry weight of the BC film,
                    which
                    was consistent with the BC thickness as visually observed (Figure 2. a, b). Intriguingly, bacteria
                    cultured in 3mL of HS medium yielded the highest amount of BC, even though the OD<sub>600</sub> of
                    bacterial
                    culture in HS was lower than that in SOC. <strong> In the end, we chose HS medium to culture and
                        product
                        BC
                        film.
                    </strong> </div>


                <image src="https://2021.igem.org/wiki/images/6/65/T--SZPT-CHINA--Results-pic-2.png" class="pic"
                    style="width: 1100px;"></image>
                <div class="tz">Figure 2. (a) Visualization of bacterial cellulose production in 2mL, 2.5mL and 3mL of
                    HS and
                    SOC medium after 4 days of fermentation in 12-well plates, (b) Quantification of the bacterial
                    cellulose
                    dry weight.

                </div>
                <div class="ej"> 3. Eletrotransformation condition exploratory experiment</div>
                <div class="pb">In order to optimize the transformation efficiency of plasmid into <i>G. hansenii</i>
                    ATCC
                    53582,
                    we conducted electroporation assay with the different voltage values, either 2.5kV or 3kV. A larger
                    amount of bacterial transformants can be found on selective plates in the 3kV-treated group.
                    Therefore,
                    we chose 3kV to perform the electroporation of <i>G. hansenii</i> ATCC 53582 with plasmids in the
                    following
                    experiment.</div>
                <div class="ej"> 4. Illumination intensity exploratory experiment</div>
                <div class="pb">



                    During the consult to the experts(<u> For more detail, please click <a
                            href="https://2021.igem.org/Team:SZPT-CHINA/Human_Practices">Human_Practices</a></u>),
                    suggestions
                    were
                    made to explore the effect of details intensity on <i>G. hansenii </i>ATCC 53582 product BC in the NIR
                    light
                    intensity in the c-di-GMP signaling and BC film production module. Then we cultured respectively
                    J23100-bphS-J23109-fcsR-pSEVA331-<i>G.hansenii</i> ATCC 53582 under 5， 10， 20， 50μW/cm<sup>2</sup>.
                    The
                    yield under 5, 10, 20, 50 μW/cm<sup>2</sup> illumination intensity had no significant difference. Therefore, we still
                    chose 50μW/cm<sup>2</sup>
                    for film production experiment.
                </div>

                <image src="https://2021.igem.org/wiki/images/1/15/T--SZPT-CHINA--result-pic-plus-plus-1.png"
                    class="pic" style="margin-left: 339px;width: 500px;"></image>
                <div class="tz">Figure 3. The BC yield at different illumination intensity</div>
                <div id="content2" class="yj">● Antipseudomonal drug production module</div>
                <div class="ej">1. Goal</div>
                <div class="pb">The function of this module is to produce a chimeric bacteriocin that can target
                    <i>Pseudomonas
                        aeruginosa </i>while having a broader antipseudomonal spectrum than pyocin S2. To enhance the
                    antipseudomonal effect, we further engineered the RBS strength to modulate the expression of SE
                    protein.
                </div>
                <div class="ej">2. What work we have done</div>
                <div class="pb">① Verify that the SE fusion protein has an antibacterial effect on <i>P.
                        aeruginosa</i>.<br>
                    ② Successfully construct a series of plasmids to express different levels of SE protein.<br>
                    ③ Through the inhibition zone test and growth curve test, screen <i>G. hansenii</i> ATCC
                    53582-derived
                    strains
                    with a pronounced antipseudomonal effect.
                </div>

                <div class="ej">3. Proof </div>
                <div class="sj">3.1 Antipseudomonal properties of SE protein</div>
                <div class="pb">IPTG was added to a final concentration of 0.1 mmol/mL to induce SE protein expression
                    in
                    <i>E.
                        coli</i> BL21 with S2-PET28A or SE-PET28A at 25℃ for 12 hours. Supernatant containing SE or S2
                    proteins were
                    obtained by sonication and centrifugation. The crude protein extract was loaded into SDS-PAGE gel
                    and
                    stained by Coomassie Blue to verify the presence of target proteins (<u> For more detailed steps,
                        please
                        click <a href="https://2021.igem.org/Team:SZPT-CHINA/Experiments">Experiments</a></u>). After
                    IPTG
                    induction, S2-PET28A-BL21 produced a 83.9 kDa
                    protein (lane 2) while
                    SE-PET28A-BL21 produced a 81 kDa protein (lane 3). In contrast, non-induced <i>E. coli</i> BL21
                    cells
                    did not
                    express S2 or SE protein (lane 1, lane 4). <strong>The results showed that the constructed plasmids
                        S2-PET28A
                        and SE-PET28A could be used to express the fusion protein </strong>(Figure 4. a).
                    <br>
                    <br>
                    Then, supernatant containing SE or S2 protein was respectively added into the culture of
                    <i>Pseudomonas
                        aeruginosa</i> PAO1, <i>P. aeruginosa</i> PAO1 Δ1150-1151（PAO1 Δ1150-1151）and <i>E. coli</i>
                    MG1655.
                    PBS
                    was used as a
                    negative control. Inhibitory effect of supernatant on the <i>P. aeruginosa</i> growth was determined
                    by
                    measuring OD<sub>600</sub> using SYNERGY H1 micro-plate reader. <strong>The result showed that the
                        SE-fusion protein could
                        prevent <i>P. aeruginosa</i> growth, irrespective of the presence of the PA1150-1151 region,
                        while
                        pyocin
                        S2
                        can only inhibit the PAO1 Δ1150-1151 mutant, but not the wild type strain. In addition, both SE
                        and
                        S2
                        proteins had no effect on <i>E. coli</i> growth</strong> (Figure 4. b).

                </div>

                <image src="https://2021.igem.org/wiki/images/6/62/T--SZPT-CHINA--Results-pic-3.png" class="pic"
                    style="margin-left: 45px;"></image>

                <div class="tz">Figure 4. (a) SDS-PAGE analysis of protein expression in engineered <i>E. coli</i>. Lane
                    1
                    S2-PET28A BL21 without IPTG induction, lane 2 supernatant of S2-PET28A BL21 with induction by
                    0.1mmoL/mL
                    IPTG, lane 3 supernatant of SE-PET28A BL21 with induction by 0.1mmoL/mL IPTG, lane 4, SE-PET28A BL21
                    without IPTG induction, (b) Optical density of bacterial culture of PAO1, PAO1 Δ1150-1151 and <i>E.
                        coli</i>
                    MG1655 treated by the supernatant of S2-PET28A-BL21 or SE-PET28A-BL21 lysate for 790 minutes. PBS
                    was
                    used as the negative control. </div>

                <div class="sj">3.2 Screen and identify an RBS to express SE protein with an optimal antipseudomonal
                    effect.
                </div>

                <div class="pb">Using high-throughput screening, we engineered the strength of RBS that control the SE
                    protein expression level in <i>E. coli</i>, and the resultant plasmids were extracted and in turn
                    introduced
                    into the <i>G. hansenii</i> ATCC 53582. <i>G. hansenii </i>ATCC 53582 culture supernatant containing
                    SE
                    proteins that
                    were expressed under control of different RBS were obtained by sonication and centrifugation and
                    further purified by infiltration with micro-pored membrane. Then, supernatant in different groups
                    was
                    individually mixed with PAO1 culture, and incubated at 30℃ for 12 hours. SYNERGY H1 micro-plate
                    reader
                    was used to measure the OD<sub>600</sub> to determine the effect on PAO1 growth.<strong> As shown in
                        Figure 5 a,
                        supernatant of culture derived from the 3<sup>rd</sup>, 4<sup>th</sup>, 7<sup>th</sup>,
                        8<sup>th</sup> colony had significant
                        antipseudomal effect.</strong>
                    <br><br>
                    3 μL of the supernatant containing SE protein was dropped on FAB plate with PAO1 evenly spread
                    <u>(for
                        more
                        detailed steps, please click <a
                            href="https://2021.igem.org/Team:SZPT-CHINA/Experiments">Experiments</a>)</u>. Then, the
                    plates
                    were incubated at 30℃
                    for 12 hours.
                    Supernatants from all the <i>G. hansenii</i> isolates have formed inhibition zone, yet with
                    different
                    sizes.
                    Isolate 3<sup>rd</sup> and 4<sup>th</sup> displayed the most significant antipseudomonal
                    effect.<strong>
                        The results of the optical
                        density measurements of PAO1 growth and the inhibition zone prove that SE protein is
                        successfully
                        expressed in the engineered <i>G. hansenii</i>. A series of <i>G. hansenii</i> containing the SE
                        expression module
                        with difference RBS were constructed to screen for the strain with the best antipseudomonal
                        property.
                        As shown in Figure 5, the 4<sup>th</sup> strain pR-RBS300-SE-J23118-IMM-pSEVA331-<i>G.
                            hansenii</i>
                        ATCC
                        53582-4# is best
                        as it has the largest inhibition zone area.</strong>
                </div>

                <image src="https://2021.igem.org/wiki/images/2/25/T--SZPT-CHINA--Results-pic-4.png" class="pic"
                    style="width:1200px;"></image>


                <div class="tz">Figure 5. (a) Optical density of PAO1 treated with the supernatants of different
                    bacterial
                    strain lysates cultured after 12 hours of growth in PBS, (b) Drop plate assay to check the
                    inhibitory
                    effect of SE proteins on <i>Pseudomonas aeruginosa</i> PAO1, (c) Strains used in Figure (b).</div>


                <div id="content3" class="yj">● C-di-GMP signaling and BC film production module</div>

                <div class="ej"> 1. Goal</div>
                <div class="pb">
                    As bacterial cellulose (BC) production in <i>G. hansenii</i> is regulated by the second messenger
                    c-di-GMP. Therefore, we aimed control BC film production by regulating c-di-GMP concentration,
                    <i>i.e</i>. so
                    that <i>G. hansenii</i> ATCC 53582 can produce BC under illumination of Near Infrared light (NIR
                    light)
                    at
                    680nm, but cannot under dark conditions.
                </div>

                <div class="ej">2. What work we have done</div>
                <div class="pb">

                    ① Construct the synthesis and hydrolysis modules of c-di-GMP in <i>G. hansenii</i> ATCC 53582.
                    <br>
                    ② Screen the best modules for BC film production, and integrate them into one single plasmid.
                </div>

                <div class="ej">3. Proof </div>
                <div class="pb">
                    We used bphS which is the coding part of the photo-activated diguanylate cyclase BphS as well as the
                    coding parts of c-di-GMP phosphodiesterase from different bacteria to construct the plasmids for
                    c-di-GMP synthesis module and c-di-GMP hydrolysis module in <i>E. coli</i> competent cells. Then the
                    plasmids
                    were extracted and transferred into the <i>G. hansenii</i> ATCC 53582.


                </div>


                <image src="https://2021.igem.org/wiki/images/d/de/T--SZPT-CHINA--Results-pic-5.png" class="pic"
                    style="width: 1200px;"></image>
                <div class="tz">Figure 6. (1) The genetic circuit for c-di-GMP synthesis module, (2) The genetic circuit
                    for
                    c-di-GMP hydrolysis module B, amplification of different regions by PCR, (c) Plasmids used in Figure
                    B.
                </div>

                <div class="pb">According to the 12-well plate test (Figure 7. A, <u>for more detailed steps, please
                        click
                        <a href="https://2021.igem.org/Team:SZPT-CHINA/Experiments"> Experiments</a></u>), BC was dried
                    and
                    weighted. A marked difference in BC production between
                    J23100-fcsR-pSEVA331-<i>G. hansenii</i> ATCC 53582 and our control group pSEVA331-<i>G. hansenii
                    </i>ATCC 53582 was
                    observed (Figure 7. B1). Therefore, J23100-fcsR-pSEVA331 was shown to have a marked hydrolytic
                    function.
                    On the other hand, J23100-bphS-bphO-pSEVA331-<i>G. hansenii</i> ATCC 53582 produced a higher level
                    of BC
                    under
                    the illumination of NIR light than the dark condition (Figure 7. B2). <strong>Based on the above
                        results, we
                        chose BphS and FcsR as the c-di-GMP synthetase and hydrolase respectively in our
                        system.</strong>
                </div>

                <image src="https://2021.igem.org/wiki/images/d/df/T--SZPT-CHINA--Results-pic-6.png" class="pic"
                    style="margin-left: 101px;"></image>
                <div class="tz">Figure 7. A. Verification experiment of BC film yield in 12-well plates B. (1) BC yield
                    by
                    J23100-fcsR-pSEVA331-<i>G. hansenii</i> ATCC 53582, J23100-yhjH-pSEVA331-<i>G. hansenii</i> ATCC
                    53582,
                    J23100-rocR-pSEVA331-<i>G. hansenii</i> ATCC 53582 and the vehicle control pSEVA331-<i>G.
                        hansenii</i>
                    ATCC 53582, (2)
                    BC yield by J23100-bphS-bphO-pSEVA331-<i>G. hansenii</i> ATCC 53582 under NIR light illumination and
                    dark
                    conditions.

                </div>


                <div class="pb">We aim to construct a photo-activated system for production of BC film in <i>G.
                        hansenii</i>
                    ATCC
                    53582, where the amount of intracellular c-di-GMP (Figure 8. A) sustained at a low level under dark
                    condition, but can reach a high level under illumination of NIR light. To this end, we constructed
                    an
                    array of plasmids by assembling different synthesis and hydrolysis components of c-di-GMP. Finally,
                    these plasmids were introduced into the <i>G. hansenii</i> ATCC 53582 via electroporation to screen
                    the
                    optimal
                    isolate in BC yield (Figure 8. B).
                    <br>
                    <br>
                    We constructed three plasmids with different promoters that can control the c-di-GMP levels. Then
                    <i>G.
                        hansenii</i> ATCC 53582 were transformed with these plasmids. Then the BC film production in
                    12-well
                    plates
                    was assessed. Among the strains we tested, a significant difference in the BC yield under different
                    illumination conditions was found for J23100-bphS-bphO-J23109-fcsR-pSEVA331-<i>G. hansenii</i> ATCC
                    53582 and
                    J23100-bphS-bphO-J23110-fcsR-pSEVA331-<i>G. hansenii</i> ATCC 53582. Notably, a background level of
                    BC
                    production was always detected in all the strains under dark conditions. <strong> In the end, we
                        chose
                        J23100-bphS-bphO-J23109-fcsR-pSEVA331-<i>G. hansenii</i> ATCC 53582 which has a significant
                        difference in the
                        BC yield under different illumination conditions as the strain relatively good matches our
                        goal.</strong>
                </div>


                <image src="https://2021.igem.org/wiki/images/1/11/T--SZPT-CHINA--Results-pic-7.png" class="pic"
                    style="width: 1200px;"></image>
                <div class="tz">
                    Figure 8. (A) The genetic circuits for c-di-GMP signal transduction and BC growth module, (B) PCR
                    identification results, lane 1 J23100-bphS-bphO-J23109-fcsR-pSEVA331-<i>G. hansenii</i> ATCC 53582,
                    lane
                    2
                    J23100-bphS-bphO -J23110-fcsR-pSEVA331-<i>G. hansenii</i> ATCC 53582, lane 3
                    J23100-bphS-bphO-J23119-fcsR-pSEVA331-<i>G. hansenii</i> ATCC 53582, (C) BC film production by
                    different
                    strains, (D) Strains used in Figure C.
                </div>

                <div id="content4" class="yj">● Safety and drug release module</div>
                <div class="ej">1. Goal</div>
                <div class="pb">
                    With the illumination of blue light, the expression of the lysis protein controlled by the pDawn
                    promoter would be activated. While the amount of the lysis proteins in the engineered bacteria
                    reaches a
                    certain threshold, the bacteria will be auto-lysed and the antipseudomonal proteins are released,
                    thus
                    antagonizing <i>P. aeruginosa</i> growth.
                </div>
                <div class="ej">2. What have we done</div>
                <div class="pb">① We have identified that the blue light-responsive promoter pDawn can activate the
                    expression of downstream genes in <i>G. hansenii</i> ATCC 53582 under blue light condition.<br>
                    ② We have constructed a set of plasmids which include pDawn to induce the expression of an
                    antiholin-free Lambda lysis cassette (S105), φX174 phage lysis gene (X174 E), and LKD16 lysis
                    cassette
                    (LKD16). After introducing these plasmids into <i>E. coli</i>, bacterial strains grow normally under
                    dark
                    conditions but do not under 470nm blue light.<br>
                    ③ The plasmids will be extracted from <i>E. coli</i> and subsequently introduced into the <i>G.
                        hansenii</i>
                    ATCC
                    53582, where the expression of X174 E can be activated under illumination of blue light
                </div>

                <div class="ej">3. Proof </div>
                <div class="sj">3.1 Functional verification of pDawn promoter in response to blue light in <i>G.
                        hansenii</i> ATCC 53582.</div>
                <div class="pb">The plasmid pDawn-RFP-pSEVA331 was firstly constructed and introduced in <i>G.
                        hansenii</i>
                    ATCC
                    53582, where its function was tested. To be specific, bacteria were cultured under either the blue
                    light
                    at 470nm or dark conditions for 24 hours, and the intensity of red fluorescent signal was detected
                    using
                    fluorescence microscope. After 24 hours of growth, <i>G. hansenii</i> ATCC 53582 in the blue light
                    treated
                    group showed red fluorescence, but no fluoresces were observed for those under dark conditions
                    (Figure
                    9). Based on these results, pDawn in <i>G. hansenii</i> ATCC 53582 can be successfully activated by
                    blue
                    light
                    irradiation.</div>



                <image src="https://2021.igem.org/wiki/images/3/38/T--SZPT-CHINA--Results-pic-8.png" class="pic"
                    style="margin-left: 125px;"></image>

                <div class="tz">Figure 9. Red fluorescence photo of the pDawn-RFP-pSEVA331-<i>G. hansenii</i> ATCC 53582
                    cultured
                    under 470-nm blue light or dark conditions for 24 hours.</div>


                <div class="sj">3.2 Functional verification of lysis protein in <i>G. hansenii</i> ATCC 53582</div>
                <div class="pb">
                    We employed the blue light-responsive promoter pDawn to control the expression of different lysis
                    genes,
                    including S105, X174 E and LKD16 (Figure 10). Random primer-guided mutagenesis on the target RBS
                    position
                    was performed (<a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>) (<u> For more detailed
                        steps, please click <a
                            href="https://2021.igem.org/Team:SZPT-CHINA/Improvement">Improvement</a></u>). To verify
                    the lysis
                    effect of these components, we conducted the plate dot assay (<u> For more detailed steps, please
                        click
                        <a href="https://2021.igem.org/Team:SZPT-CHINA/Experiments">Experiments</a></u>). Expression of
                    lysis genes
                    will be considered effective when bacterial
                    colonies can grow
                    normally in the dark, but not under blue light irradiation.
                </div>

                <image src="https://2021.igem.org/wiki/images/0/08/T--SZPT-CHINA--Results-pic-9.png" class="pic"
                    style="margin-left: 113px;"> </image>
                <div class="tz">Figure 10. Genetic circuit for safety and drug release module. </div>



                <div class="pb">
                    To be specific, 20 single colonies on the culture plate were picked to spot on two parallel plates
                    (if
                    the number of single colonies on the culture plate is less than 20, picked all of them). The plates
                    were
                    incubated either in the dark or under blue light illumination for 24 hours to observe the colony
                    morphology, based on which we determine whether there is a lysis effect.
                    <br><br>
                    Although the colony of S105 could not grow under blue light its growth state was abnormal in the
                    dark.
                    We attributed this phenomenon to a basal expression level of lysis protein controlled by pDawn
                    promoter
                    (Figure 11. A (a1), (a2)). Of note, the 4<sup>th</sup> isolate of with pDawn-X174 E-pSEVA331-
                    <i>E. coli</i> grew
                    normally in
                    the dark but not under the blue light irradiation（Figure 11. A (b1) and (b2)）. Additionally, as
                    shown in
                    Figure 11. A (c1) and (c2), all the colonies of <i>E. coli</i> with pDawn-LKD16-pSEVA331- <i>E.
                        coli</i>
                    were
                    able to
                    grow under both conditions, whereas the growth of 4<sup>th</sup>, 9<sup>th</sup>,10<sup>th</sup> and
                    12<sup>th</sup> colonies was remarkably
                    inferior than those in the dark, indicating that pDawn-LKD16 allowed bacteria to be lysed in
                    response to
                    blue light, yet with a modest effect.
                    <br><br>
                    In order to construct <i>G. hansenii</i> ATCC 53582 strains with the capability of light-responsive
                    cell
                    lysis,
                    we firstly introduced the plasmids that have lysis effect in <i>E. coli</i> into the <i>G.
                        hansenii</i>
                    ATCC
                    53582,
                    and then 8 single colonies were picked to perform the drop plate assay. Two copies of plates were
                    separately incubated under the blue light and dark conditions at 30℃. The bacterial colony
                    morphology,
                    including the size and thickness, was observed every 24 hours to determine the lysis effect. After
                    introducing the plasmid containing S105, X174 E or LKD16 into the <i>G. hansenii</i> ATCC 53582,
                    they can
                    cause
                    similar colony-lysis phenotype. While the plasmids containing pDawn-S105 completely inhibited growth
                    of
                    <i>G. hansenii</i> ATCC 53582 under both conditions. (Figure 11. B (a), (b) and (c)). We thus
                    preserved
                    the
                    pDawn-X174 E-pSEVA331-<i>G. hansenii</i> ATCC 53582 and pDawn-LKD16- pSEVA331-<i>G. hansenii</i>
                    ATCC
                    53582 . And these
                    strains were spotted on the culture plates for screening. The strain pDawn-X174 E-pSEVA331-<i>G.
                        hansenii</i>
                    ATCC 53582-7# showed a stable lysis effect, while the others did not. (Figure 11. C). <strong>In the
                        end, we
                        chose pDawn-X174 E-pSEVA331-<i>G. hansenii</i> ATCC 53582-7# which can be activated under
                        illumination
                        of blue
                        light as the goal strain.</strong>


                </div>

                <image src="https://2021.igem.org/wiki/images/9/9c/T--SZPT-CHINA--Results-pic-10.png" class="pic"
                    style="margin-left: 115px;">

                </image>
                <div class="tz">Figure 11. Drop plate assay to assess the inducible production the lysis protein by
                    470-nm
                    blue light illumination, (A) <i>E. coli </i>DH5α transformed with different lysis proteins, (B)
                    <i>G.
                        hansenii</i> ATCC
                    53582 transformed with different lysis proteins, (C) pDawn-X174 E-pSEVA331-<i>G. hansenii</i> ATCC
                    53582,
                    which
                    stably showed the lysis effect, (a1) and (a2), pDawn-S105-pSEVA331, (b1) and (b2), pDawn-X174
                    E-pSEVA331,
                    (C1) and (C2), pDawn-LKD16-pSEVA331,(a1), (b1) and (C1) are incubated under 470-nm blue light, (a2),
                    (b2) and (C2) are incubated under dark conditions.
                </div>
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